The primary aim of the proposed research is to study the role of Akt (protein kinase B) in epithelial carcinogenesis. During the previous funding period, we obtained evidence for a role of insulin-like growth factor 1 (IGF-1)/IGF-1R signaling in multistage mouse skin carcinogenesis. As a result, we generated transgenic mice that overexpress human IGF-1 in the basal layer of the epidermis using the bovine keratin 5 (BK5) promoter. These mice (referred to as BK5.IGF-1) exhibit a skin phenotype consisting of chronic hyperplasia with an increased labeling index [measured by bromodeoxyuridine (BrdU) incorporation]. These mice, if initiated with 7,12-dimethylbenz[a]anthracene (DMBA), develop papillomas indicating that enhanced IGF-1R signaling can act as a skin tumor promoter. Furthermore, BK5.IGF-1 mice are hypersensitive to the skin tumor promoting actions of the phorbol ester 12-0- tetradecanoylphorbol-13-acetate (TPA). We have also found that phosphatidylinositol-3 kinase (PI3K) and Akt activities are elevated in epidermis of BK5.IGF-1 mice and that both glycogen synthase 3beta (GSK3beta) and Bad are hyperphosphorylated on serine residues. Therefore, activation of Akt in epidermis of BK5.IGF-1 mice leads to alterations in downstream signaling pathways. In addition, we found that the PI3K inhibitor LY294002 effectively blocks IGF-1 mediated skin tumor promotion whereas the mitogen-activated protein kinase (MAPK) inhibitor, PD98059, does not. In contrast, PD98059 blocked TPA promotion in nontransgenic mice while LY294002 had little or no effect. However, in more recent experiments we have made the novel discovery that diverse tumor promoters, including TPA, okadaic acid (OA) and chrysarobin (CHRY) upregulate Akt kinase activity in epidermis of nontransgenic mice very early after single topical applications. Collectively, these data raise the exciting possibility that activation of Akt is an important early event for skin tumor promotion and that multiple mechanisms may be involved in the activation of Akt by skin tumor promoters. In this proposal, we will test the hypothesis that activation of Akt is critical for the skin tumor promoting action of diverse skin tumor promoters including TPA, OA, CHRY and IGF-1. The proposed research will lead to a better understanding of the role of Akt signaling in the context of multistage carcinogenesis and more specifically of the mechanism(s) involved in skin tumor promotion in an in vivo model of epithelial carcinogenesis. In addition, successful completion of the proposed studies could lead to the identification of Akt as an important target for future chemoprevention studies. The Specific Aims are i) To further characterize the alterations in Akt and its downstream signaling pathways in epidermis of tumor promoter-treated mice; ii) To determine the mechanism(s) for activation of Akt in epidermis by diverse skin tumor promoters; iii) To determine the impact of elevated Akt activity on epidermal proliferation/differentiation and on skin tumor promotion; and iv) To determine the impact of Akt deficiency on epidermal proliferation/differentiation and on skin tumor promotion. [unreadable] [unreadable] [unreadable]